Immortalization of dendritic cells with v-myc oncogene

ABSTRACT

The present invention refers to immortalized dendritic cells, to a process for their production from primary cultures and to their use for the activation, in vivo or in vitro, of T lymphocytes in antigen specific way.

This application is a Continuation of U.S. application Ser. No.08/549,666, filed Nov. 29, 1995, U.S. Pat. No. 5,856,180; which is aNational Stage application of PCT/EP94/01720, filed May 26, 1994, whichclaims priority to Italian application MI93A001118.

The present invention refers to immortalized dendritic cells, to aprocess for their production from primary cultures and to their use forthe activation, in an vivo or an in vitro, of T lymphocytes in anantigen specific way.

The antigen specific immune response is the result of interactionsbetween T, B lymphocytes and antigen presenting cells (APCs). The typeof immune response elicited by the antigen (cell-mediated cytotoxic orhumoral responses) and the generation of immune memory are influenced bythe interaction between these cells and their products, by the sitewhere these interactions occur and by the nature of the antigen itself.It is believed that activation and suppression of the immune system arealso the results of the above mentioned variables which, if notregulated, can lead to autoimmune diseases and tolerance induction.

Dendritic cells (DC), first described by Steinman and Cohn in 1973 (J.Exp. Med. 137:1142, 1973), are a population of widely distributedleukocytes that play a key role in the immune system (Steinman R. M.1991, Annu. Rev. Immunol. 9:271-296; Romani N. et al., 1992, SpingerSemin. Immunopathol. 13:265) given that they are: i. highly specializedin antigen presentation, ii. the principal activators of resting T cellsin vitro (Inaba M. D. et al., 1984, J. Exp. Med. 160:858; Croft M. etal., 1992, J. Exp. Med. 176:1431), iii. the major source of immunogenicepitopes for specific T cell clones following administration of antigenin vivo (Inaba K. et al., 1990, J. Exp. Med. 172:631; Crowley M. et al.,1990, J. Exp. Med. 172:383) and iiii. the most potent initiators ofprimary T cell-mediated responses in vivo (Lechler R. I. et al., 1982,J. Exp. Med. 155:31).

Several studies have suggested that DC provide naive T cells with allthe necessary signals required for activation and proliferation(Steinman R. M. and Romani N., above cited). These signals are generatedby the interaction of complexes of major histocompatibility complex(MHC) molecules and antigenic peptides with the T cell receptor (DavisM. et al., 1988, Nature 334:395), and by the engagement ofco-stimulatory molecules, including binding of B7/BB1 molecules onantigen presenting cells (APC) to CD28 receptor on the T cell surface(Young J. W. et al., 1992, J. Clin. Invest. 90:229; Nabavi N. et al.,1992, Nature 360:266). The first signal alone elicits effector functionsonly in activated T cells and is unable to stimulate naive or resting Tcells, which in the absence of co-stimulatory signals can enter a periodof unresponsiveness (Inaba K. et al., 1985, Science 229:475; Mueller D.L. et al., 1989, J. Immunol. 142:2617; Tan P. et al., 1993, J. Exp. Med.177:165). The expression of the co-stimulatory molecule B7/BB1 on DCpopulations has been recently reported and shown to be critical inDC-driven primary T cell responses (Larsen C. P. et al., 1992, J. Exp.Med. 176:1; Symington F. W. et al., 1993, J. Immunol. 150:1286; Liu Y.et al, 1992, Eur. J. Immunol. 22:2855).

Understanding the mechanisms underlying the potent stimulatorycapacities of DC could explain how T cells are primed, and how theimmune response is initiated. With this knowledge one might try tomanipulate immune responses at very early stages and provide a way forinducing immunity or tolerance. However, an important limitation in thestudy of DC biology has been the small numbers of cells available fromany tissue, given that no stable cell lines that are clearly similar toDC have been obtained so far.

Three different tissues have been used as major sources of DC: mousespleen, the epidermis, where DC are known as Langerhans cells, and humanblood. In each case DC constitute a tiny fraction of the startingtissue, representing about 1% of crude spleen (Steinman R. M. et al.,1979, J. Exp. Med. 149:1) or epidermal (Schuler G. et al., 1985, J. Exp.Med. 161:526; Romani N. et al., 1989, J. Invest. Dermatol. 93:600) cellsuspensions and 0.1-1% of peripheral blood mononuclear cells(Freudenthal P. S. et al., 1990, Proc. Natl. Acad. Sci. USA 87:7698).More recently, Inaba and co-workers have described a method forgenerating DC from both peripheral blood and bone marrow precursors, butcell proliferation ceases within 1-3 weeks (1992. J. Exp. Med.175:1157).

A different approach is to generate cell lines of DC from primarycultures and identify a method which will allow the immortalization ofDC. At the present time, however, there is a need for an effectivemethod of introducing genetic material into DC and enable them toexpress genetic material which they do not usually express.

Unsuccessfull attempts in this respect have been carried out byKomatsubare et al. (Microbiol. Immunol. 32(8), 869-875, 1988) whoinserted V-SRC and Ha-RAS oncogenes in murine dendritic cells:immortalized dendritic cells did not show however characteristics ofdendritic cells.

DISCLOSURE OF THE INVENTION

It has now been found a method to obtain cell lines with the phenotypicand functional characteristics of leukocytes able to present antigenicpeptides to cells of the immune system and induce specific immuneresponses in vivo and in vitro. In particular, the invention concernsthe immortalization of antigen presenting cells (APC) such as thedendritic cells (DC) by introduction of exogenous genetic material, notexpressed normally by these cells, able to generate indefinite cellgrowth (immortalization) in the transfected or infected cells withoutaffecting their APC function.

The immortalized APC according to the invention have inserted andexpress the exogenous genetic material.

The genetic material able to immortalize APC and in particular DC, iseither:

1) RNA or DNA which can be normally present in APC but not expressed tolevels which could be of biological significance;

2) RNA or DNA normally present in APC but not expressed to levels whichcould be of biological significance, modified in order to be expressedin APC and in particular in DC;

3) RNA or DNA not normally present in APC and in particular in DC orcombinations therefrom useful for their expression in APC and inparticular in DC.

The genetic material able to immortalize APC and in particular DC canbe:

a virus or a retrovirus or a complex of viruses or retroviruses or partof them obtained from any kind of species;

any RNA or DNA or combinations of them obtained from viruses orretroviruses or from their recombination;

any modified (either spontaneously or engineered) RNA or DNA of viral orretroviral origin or their combination.

The genetic material (any RNA, DNA or combinations or modifications ofthem) can be inserted in:

vectors;

expression vectors;

DNA different from the original viral or retroviral genomes;

vectors containing genetic markers for selection of the cells in whichthe genetic material has been inserted.

In general, the specific order in which the different elements areassembled together is not critical, so that the flanking regions mightbe first bound to a replication system including a marker or otherregions, such as enhancers, transcriptional regulatory regions, or thelike, prior to insertion of the gene. The process in which the variousfragments are brought together will depend on a number or factorsrelated to ease of construction, choice of restriction sites, use ofselection methods, availability of particular fragments, and the like,which ultimately are within the choice capability of the man skilled inthe art.

The genetic material allows the immortalization of APC directly fromprimary cultures in which DC may represent a minor population, withoutany need of particular selection methods.

In particular, the exogenous genetic material allowing the achievementof the above cited results derive from retroviral vectors containing thev-myc oncogene. Preferably, the retroviral vector is obtained byco-transfection of two retroviruses, at least one of them containing thev-myc oncogene.

According to a specific embodiment of the invention, the exogenousgenetic material contains a retroviral oncogene of avian origin fusedwith a coding retroviral sequence of mouse origin; in particular, theavian oncogene is a mutated v-myc gene fused with part of the envprotein derived from avian retrovirus MH₂ (T. Graf et al., Biochim.Biophys. Acta, 516, 269-299, 1982) and from the murine AKR retrovirus(R. Risser et al., Ann. Rev. Genet., 17, 85-121, 1983) and in "RNA TumorViruses", R. Weiss et al., 2nd ed. Cold Spring Harbor, 1985. Moreparticularly, as shown in FIG. 1a, the MH₂ and AKR retroviruses areco-transfected into murine macrophages as disclosed by Righi et al.,Oncogene, 4, 223-230, 1989. A cell line is obtained which produces aretrovirus complex (3RV) which can immortalize cells of the same type(Righi et al., Eur. J. Immunol. 19, 1443-1448).

By infecting brain macrophages with this retrovirus complex, the cellline N11 is obtained, which in turn produces the virus VN11 (Righi etal., Oncogene, 6, 103-111, 1991) which is able to immortalizemacrophages (L. Pirami et al., Proc. Natl. Acad. Sci. USA 88, 7543-7547,1991). The N11 cell line was deposited according to the Budapest Treatyat the European Collection of Animal Cell Cultures, Salisbury, UK, onDec. 5, 1993, under the accession number 93051207.

The VN11 virus may be used to directly immortalize dendritic cells or itmay be cloned into a vector able to transfect "packaging" cell linesproducing retroviral vectors which are in turn able to transfectdendritic cells.

The transfection of the latter may be obtained by co-cultivatingdendritic cells with "packaging" cells. Examples of "packaging" celllines which may be used according to this invention are the psi2 cellline, disclosed in "Experimental Manipulation of gene expression" M.Inouy (ed.), 155-173, 1983 and in R. Hann et al. Cell, 33, 153-159,1983; an equivalent derivative of psi2 cell line, named psi am, isdisclosed by R. Clone et al., Proc, Natl. Acad. Sci. USA, 81, 0349-6353and deposited at the American Type Culture Collection (Rockville,Md.-USA; accession number CRL 8859).

The genetic material preferably used for transfection, according to theinvention, contains the mutated v-mycMH2 gene fused with part of theenvAKR mouse retroviral sequence (EMBL Database entry Accession n. Z26309).

The described method of immortalization of APC which uses thisparticular recombinant oncogene can be extended to any other cellular orretroviral oncogenes.

Because genes can be introduced into DC using a retroviral vector, theycan be "on" (subject to) the retroviral vector control; in such a case,the gene of interest is transcribed from a retroviral promoter. Apromoter is described as a specific nucleotide sequence recognized by anumber of transcription factors in order to allow RNA polymerasecomplexes to initiate the RNA synthesis. Retroviral vectors can bedesigned in order to have other promoter elements (in addition to thepromoter of the recombinant retrovirus) which are responsible for thetranscription of the gene. For example, it is possible to modify thevector inserting an additional promoter modulated by external factorsallowing to control the level of polypeptide being produced by the DC byadding to the culture that external factors.

Incorporation of this promoter or another promoter influenced byexternal cues also makes it possible to regulate the production of thepolypeptide by the engineered DC.

The cell lines obtained with the introduction of the exogenous geneticmaterial and which are part of the invention, have in addition to otherproperties, the characteristics of APC and the capacity to activatecells of the immune system such as T lymphocytes.

Preferred APC are DC. According to the above description, the exogenousgenetic material has been introduced in DC; for the purposes of theinvention DC comprise DC from bone marrow, peripheral blood, cord blood,epidermis (Langerhans cells), follicular and interstitial DC fromgerminal centers and interdigitating DC of the lymphoid organs. Thetissue origin is not a limitation for the present invention.

The genetic material introduced in these cells confers to DCimmortalization (unlimited cell growth) and can be modified in such away that the product is changed and/or its expression can be eitherconstitutive or inducible after activation. The product of such geneticmaterial can be any polypeptide, secreted or anchored in the cellmembrane, such as an antigen or related peptide, a lymphokine ormembrane protein.

In addition, the genetic material described above can also be introducedin other cell types able to activate the immune system, such as, forinstance, macrophages or into cells normally unable to activate theimmune system which have been modified or induced to obtain such effect.

The invention also refers to a method to induce the immune system of anorganism to generate a specific, predetermined immune response using thecell lines which are the object of the invention.

The cell lines which are the object of the invention can be loaded invitro with any kind of antigen and then be used with the followingadvantages:

1) they can activate T lymphocytes ex vivo. Co-culture of naive orantigen-specific T lymphocytes with the antigen-loaded DC cell line (asantigen presenting cells) can result in priming and expansion of theantigen-specific T lymphocytes. This will be of particular interest forthose antigens which either do not generate in vivo a protective immuneresponse (for instance tumor antigens or pathogens such as viruses,fungi and intracellular parasites) or could not be used in vivo fortheir undesirable side effects. Antigen-specific T lymphocytes activatedand expanded in vitro using DC cell lines, can be reintroduced in theorganism of origin (adoptive transfer) and generate the desired immuneresponse.

2) They can be used directly in vivo to activate the desired hostantigen-specific T lymphocytes. This type of cell vaccination could beextremely useful when the introduction of the antigen into the organismis not desirable or when the pathogen is already present into theorganism or when the processed antigen (peptide-MHC complex) is moresuitable for vaccination compared to the whole soluble pathogen.

3) They can generate immunological memory since they can prime virgin Tcells as well as induce memory T cells in vivo; this is particularlyrelevant in cell vaccination. In this regard, they can be considered asnatural adjuvants.

4) They can be loaded with antigens which could be associated witheither class I or class II MHC molecules depending on the type of immuneresponse desired. In fact, genes coding for antigenic determinants ofpathogens such as viruses or intracellular bacteria can be transfectedinto DC cell lines and their products will be primarily associated withclass I MHC molecules; conversely DC cell lines could also be loadedwith purified or recombinant proteins from pathogens such as viruses orintracellular bacteria and primarily associated with class II MHCmolecules; in this way it is possible, depending on the type of loadingof DC cell lines, to direct in vivo the immune response (cell-mediatedor antibody mediated) by injecting either type of pre-loaded DC cellline.

5) They can be used to induce antigen specific immune responses towardantigens that depend on non MHC class I or class II antigens, such as inthe case of the CD1 family.

6) They can be used to induce specific T Helper cell subsets, such asthe TH1 or TH2 subsets, in those cases in which the immune system is notable to develope the desired response.

7) They can be modified with genes which can express products able tomodulate the immune response, such as cytokine genes. These genes can beunder the control of promoter/enhancer elements which may be inducedthrough specific signaling pathways following the interaction betweenmembrane receptors and ligands.

8) They can be modified in order to render them not viable whenintroduced in human beings by rendering them, as an example, dependingon a factor or a culture condition or a molecule that is not normallypresent in mammals.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C

FIG. 1a: preparation scheme of the retroviral vector MIB-psi2-N11.

FIG. 1b: partial restriction map of the retroviral vector MIB-psi-N11and Northern blot analysis of CB1 cell line of the invention using aspecific probe for the avian gene v-myc-MH2:

A) CB1 cells;

B) positive control.

Viral transcripts of genomic and subgenomic size are show by the arrows.

FIGS. 2A-2J:

a) FACS analysis of D2SC/1 dendritic cell clones using a panel ofantibodies and compared with fresh dendritic cells as reported in theliterature.

b) Immunohistochemical analysis of CB1 cells using 2A1 (A) and M342(a)antibodies and compared with she relative controls, respectively (B) and(D).

FIG. 3:

Growth curve of an immortalized DC clone.

FIGS. 4A & B:

a) MLR assay;

b) antigen specific T-lymphocyte activation assay.

FIGS. 5A & B:

Contact sensitivity (CS) induction by CB1 cells modified with FITC(a) orDNBS(b).

DETAILED DESCRIPTION OF THE INVENTION

From primary tissue cultures in which APC represent only a minorfraction of the starting material, it is possible to transduce into APCand in particular into DCs (for the purposes of the invention, DCscomprise DC from bone marrow, peripheral blood, cord blood, epidermis(Langerhans cells), follicular and interstitial DC from germinal centersand interdigitating DC of the lymphoid organs) exogenous geneticmaterial able to generate continuous cell lines of functional APC and inparticular of functional DCs. Primary cultures can be obtained fromspleen cell suspensions. The cultures are typically heterogeneousmixtures of many cell types, each of which can be induced to replicate alimited number of generations. Primary cells are thus distinguishablefrom cell lines, which have been immortalized as a result of anancestral transformation event or other mutation resulting in continuousor indefinite growth in culture. After about three weeks and without anykind of selection it is possible to isolate proliferating cells andestablish continuous cell lines which can be further cloned.

The immortalization is achieved with genetic material consisting of anRNA or DNA construct which, in order to provide transforming capability,incorporates at least one oncogene, which can be derived from viral orcellular genomes or mammalian or avian chromosomal DNA. In general,infection of an appropriate target cell with an acutely oncogenicretrovirus leads to oncogenic transformation. Although the mechanismsleading to oncogenesis are not clearly understood, numerous viraloncogenes or v-oncs, as well as their cellular homologues, known asproto-oncogenes or c-oncs, have been catalogued. Several mechanisms havebeen proposed by which c-oncs acquire transforming capabilities,including control by strong viral promoters, gene copy amplification,addition of viral enhancer sequences, rearrangement, and mutation. Forpurposes of the present invention oncogenes include any genomic materialor materials substantially homologous to oncogenic sequences which arecapable of transforming a primary APC. As an example of potentialtransforming genomic materials lists of oncogenes are provided by Bishopet al., in Weiss et al., eds., RNA Tumor Viruses, Volume 1 (Cold SpringHarbor Laboratory, N.Y. 1984) pp 1004-1005, and Watson et al., MolecularBiology of the Gene, 4th Ed., Vol. II, (Benjamin Cummings, Menlo Park,Calif.) pp 1037. Included are the known oncogenes such as src, yes, abl,fps, fes, erbB, fms, ros, kit, mos, raf, H-ras, K-ras, sis, myc, myb,fos, ski, and erbA. Several oncogene products appear to be homologues ofgrowth factors, growth factor receptors, or are nuclear proteins whoseeffects can be mimicked by the addition of their products to the cellculture. Many oncogenes can be obtained from public collections ofdeposited biological materials.

A combination of various retroviruses, including hybrid retrovirusesgenerated by spontaneous recombination in vitro or retroviruses in whichgenes have been fused and fusion products generated, can also be usedfor the immortalization of cells. These events can be obtained byinfection of myeloid cells with retroviruses according to the describedprocedures (Righi et al., 1989, Oncogene: 4, 233-230; Blasi E. et al.,Nature: 318, 667-669 (1985)) and according the mechanisms described in:"Weiss et al. eds. RNA tumor viruses, Cold Spring Harbor Laboratory,1985.

A limitation of these procedures is represented by the fact that thetransduced genetic material is not characterized and once introducedinto the target cell could generate a viral progeny. These are undesiredfeatures particularly when the obtained cell lines have to be injectedinto an organism.

This invention can overcome these problems: in particular, one of theprovirus able to immortalize DC cells in vitro has been molecularlycloned in a lambda phage. The insert, containing the whole viral genomeplus the flanking regions derived from the mouse cell from which theprovirus was originally cloned, was then subcloned in a pUC18. Usefulplasmid vectors for amplifying the retroviral genetic elements inbacterial hosts prior to transfection are constructed by inserting aretroviral DNA sequence encoding the elements described previously in avector that could include one or more phenotypic selectable markers andan origin of replication for bacterial hosts, such as E. coli., althoughothers may also be employed as a matter of choice. Thus, a usefulmammalian/bacterial shuttle vector can comprise a selectable marker andbacterial origin of replication derived from commercially availableplasmids comprising genetic elements of the well known cloning vectorpBR322 (ATCC 37017). The resulting recombinant retrovirus would thus becapable of integration into the chromosomal DNA of an infected hostcell, but once integrated, be incapable of replication to provideinfective virus, unless the cell in which it is introduced containsanother proviral insert encoding functionally active trans-acting viralproteins. The plasmid p316 was transfected into psi cells (packagingcell line) [Mann et al., Cell. 33:153 (1983)] which confer a limitedecotropism. The transfection can be carried out by any convenient meanssuch as micro injection, DEAE-dextran mediated transfection, calciumphosphate precipitated DNA transfection and electroporation (seeBonerji, J. et al., Cell 33, 729-740 (1983); Graham F. L. and Van der EbA. J., Virology, 52, 456-467 (1983) and Potter H. et al., Proc. Natl.Acad. Sci. USA, 81, 7161-7165 (1984)). The viral particles produced bythe transfected packaging cell line can infect DC with a limited tropisminducing their in vitro proliferation and allowing the isolation of celllines from these cell derivatives.

The infected DC are immortalized by these defective retroviruses butwill not propagate them any further. Retroviruses to be adapted for usein accordance with this invention can be derived from many avian ormammalian hosts. However, a requirement for use is that the virus becapable of infecting cells which are to be the recipients of the newgenetic material to be transduced using the retroviral vectors. Examplesof retroviruses include avian retroviruses such as avianerythroblastosis virus (AMV), avian leukosis virus (ALV), avianmyeloblastosis virus (ABV), avian sarcoma virus (ASV), Fujinami sarcomavirus (FuSV), spleen necrosis virus (SNV), and Rous sarcoma virus (RSV);bovine leukemia virus (BLV); feline retroviruses such as feline leukemiavirus (FeLV) or feline sarcoma virus (FeSV); murine retroviruses such asmurine leukemia virus (MuLV), mouse mammary tumor virus (MMTV), andmurine sarcoma virus (MSV); rat sarcoma virus (RaSV); and primateretroviruses such as human T-cell lymphotropic viruses 1 and 2(HTLV-1,2), and simian sarcoma virus (SSV). Many other suitableretroviruses are known to those skilled in the art. A taxonomy ofretroviruses is provided by Teich, in Weiss et al., eds., RNA TumorViruses, 2d ed., Vol. 2 (Cold Spring Harbor Laboratory, New York, 1985)pp 1-16. The limited ecotropism of the retroviruses can be overcomeusing the cloned viral genome which can be transfected into the targetcells by electroporation, protoplast cell fusion or calcium phosphateprecipitation.

For example, the cell lines which are the object of the presentinvention can be obtained by infection of unselected DC with the mutatedavian oncogene v-mycMH₂ (Graf T., Biochim. Biophys. Acta, 516, 269-299,1982) fused with part of the env AKR mouse retroviral sequence (RisserR. et al., Ann. Rev. Genet. 17, 85-121, 1983). This sequence is found inthe macrophage-derived cell line N11, producing the VN11 virus (Righi etal., Oncogene, 6, 103-111, 1991) which is able to immortalizemacrophages (Pirami L. et al., Proc. Natl. Acad. Sci. USA, 88,7543-7547, 1991). The cell line N11 has been deposited according to theBudapest Treaty at the European Collection of Animal Cell Cultures,Salisbury, GB, on May 12, 1993 under the accession number 93051207.

The VN11 virus may be directly used to immortalize dendritic cell or itmay be cloned into a vector which may be transfected into "packaging"cell lines.

Dendritic cells may then be infected by co-cultivation with said"packaging" cells, or by supernatants from these packaging cells.

For instance, a retroviral vector, named MIB-psi2 N11, was obtainedtransfecting into-the psi cell line the genome of VN11 virus, clonedinto a suitable plasmid vector, together with the neo gene, derived fromthe TN5 transposon.

The plasmid vector may be obtained according to conventional method,e.g. by inserting Hind III-EcoRI fragments of a DNA library of N11 cellsinto pUC18, transformation of HB101 E. coli cells with the obtainedconstructs and subsequent screening by hybridization with a labelledprobe derived from a fragment of the myc-MH2 gene.

DC can be either infected with MIB-psi2 N11 retroviral vector or theycan be co-cultivated with the viral producer psi cells (psi2-N11). As anexample of the co-culture procedure, psi line producing the virus can betreated with mitomycin C at 5 micrograms per ml at about 37° for abouttwo hours in RPMI 1640 without serum. psi cells were treated withmitomycin C on a 10 centimeter dish and washed several times with DME.The suspension of spleen cells was then seeded onto this dish and leftfor about 24 hours spleen cells are then recovered by gentle pipettingand replated into a different petri dish.

Retroviral Vectors

Details of the construction of a retroviral vector are contained inMulligan R. C., Construction of Highly Transmissable Mammalian CloningVehicles Derived from Murine Retroviruses, In: Experimental Manipulationof Gene Expression, M. Inouye (ed), 155-173 (1983); Mann R. et al.,Cell, 33: 153-159 (1983); Williams D. A. et al., Nature, 310: 476-480(1984).

The teachings of these publications are incorporated herein byreference.

The psi2 cell line described by Mulligan and co-workers was created bytransfecting NIH 3T3 fibroblasts with pMOV-psi-, which is an ecotropicMoloney murine leukemia virus (Mo-MuLV) clone. pMOV-psi- expresses allthe viral gene products but a sequence necessary for encapsidation ofthe viral genome. Moreover, pMOV-psi expresses an ecotropic viralenvelope glycoprotein which recognizes a receptor present only on mouse(and closely related rodent) cells.

Another cell line is the NIH Psi am a modified pMov-Psi-genome in whichthe ecotropic envelope glycoprotein has been replaced with envelopesequences derived from the amphotropic virus 4070A yelding a cell lineproducing a recombinant virus with wider amphotropic host range.

In addition, the plasmid containing the viral genome can be modifiedprior to transfection into the packaging psi cell line with exogenousgenetic material from other species and then introduced into DC. Theintroduced genetic material can be a genetic marker or selection genescoding for intracellular, membrane bound or secreted polypeptides thatwould further allow the identification and selection of the immortalizedDC cells. The genetic material can also contain regulatory sequences orsequences able to stabilize mRNA products, such as the 3' untranslatedregions of some messenger RNAs or intron regions. The provided dominantselectable marker can be, for example, any antibiotic resistancephenotype such as neo (G418 resistance), hygro (hygromycin resistance),or gpt (mycophenolic acid resistance) genes that can be used providedthat a suitable donor is selected as source of the DC to beimmortalized; such selectable markers, are widely available amongresearchers. An additional useful genomic material to be inserted intothe vector is any gene product having value or utility that depends uponthe environment in which it is translated, for example viral, bacterialor tumor cell antigens, or biologically active molecules such as antigenprocessing polypeptides, cytokines, hormones, growth factors as well astheir receptors or homologues of the foregoing or any polypeptides thathas a tissue specificity.

In particular genes coding for protein antigens or peptides derived fromthese antigens, as well as secreted lymphokines or membrane proteinssuch as MHC polypeptides, can also be introduced in the originalMIBpsi-2-N11 vector.

Using the MIBpsi2-N11 vector several DC clones have been immortalizedand two of them named CB1 and D2SC/1 extensively characterized andreported as an example. In one embodiment of this invention, thefibroblast cell line used as the feeder layer for (in cocultivationwith) DC is a Psi am line producing the VN11 virus.

Spleen cell suspensions from newborn DBA/2 (CB1 cells) or BALB/C (D2SC/1cells) mice were infected with the retroviral vector MIBpsi2-N11 andabout two to four weeks after infection, foci were observed andproliferating cells, detaching from adherent aggregates, were cloned;characteristically, these cells display sheet-like processes, with astriking motility which is not exhibited by other leukocytes. Theestimated doubling time of the immortalized cell lines is about 20 hrs.

Two clones, named CB1 and D2SC/1 have been characterized more in detailalso by means of antibodies specific for surface or intracellularmarkers.

In the case of lines derived from dendritic cells the antibodies N418recognizing the CD11c molecule (Metlay et al., J. Exp. Med. 171,1753-1771 (1990)) M342 (Agger et al., J. Leukoc. Biol. 52, 34-42 (1992))and 2A1 (Inaba et al., J. Exp. Med. 175, 1157-1167 3o (1992)) may beused. The use of those antibodies as well as many other recognizingother specific markers show that CD lines have many of thecharacteristics reported for Langerhans cells (De Paufilis et al., J.Invest. Dermatol. 93, 60-69 (1989)) and for DC (Inaba K. et al., J. Exp.Med. 176, 1963-1702 (1992)).

The stimulatory activity of DC is partially connected with the presenceof the membrane protein B7/BB1 which has been identified on several APCtypes (Linsley P. S. et al, J. Exp. Med. 173, 721-730 (1991); Linsley P.S. et al., Science 257, 792-795 (1992)) and is considered essential forthe induction of virgin T lymphocytes proliferation in MLR assays (MixedLymphocyte Reaction) and in inducing an antigen specific proliferativeresponse in lymphocytes expressing the CD4 marker (Larsen C. P. et al,J. Exp. Med. 176, 1215-1220 (1992)). It is possible, for instance, tomeasure the expression of B7/BB1 by means of flow cytofluorimetry. Theresult shows that in the DC clones the B7/BB1 gene is constitutivelyexpressed.

The stimulatory activity of DC in a primary MLR (Steinman R. M. et al.,J. Exp. Med. 157, 613-617 (1983)) is a typical characteristic of thesecells. The CB1 cell line has a stimulating activity in inducing aprimary proliferative response in lymphocytes in vitro in an allogenicreaction of MLR type.

A further functional characteristic of DC consists in the capacity ofstimulating in an antigen-specific way both T lymphocytes activated bythe first contact with the antigen and virgin T lymphocyts (Romani N. etal., J. Exp. Med. 169, 1169-1178 (1989); de Brujin M. L. H. et al., Eur.J. Immunol. 22, 2347-2352 (1992); Croft M., J. Exp. Med. 1765, 1431-1437(1992)).

The cell lines of the invention may be used to induce in vivo a primaryantigen-specific response and without the adjuvant's help. The DC may beadministered in vivo after in vitro exposure to the antigen.

They are able to process and present a native antigen such as myoglobinof whale sperm, to a T-cell clone specific for this antigen.

The DCs are also able to induce cell-mediated responses such as contactsensitivity (Sullivan S. et al., Immunol. 137, 2460-2467 (1986)), thegraft rejection of allogenic transplants (Lechler R. I. et al., J. Exp.Med. 155, 31 (1982); Larsen C. P. et al., J. Exp. Med. 172, 1483(1990)), the activation of MHC restricted T-lymphocytes (Inaba K. etal., 172, 631 (1990)) and T-lymphocytes mediated antibodies (Sornasse etal., J. Exp. Med 175, 15-21 (1992)).

The contact sensitivity (CS) is a particular kind of delayed immuneresponse occurring when the organism is immunized at the epicutaneouslevel and subsequently receives a stimulation with the reactive hapten(Sullivan S. et al., J. Immunol. 137, 2460-2467). It is possible toevidence CS even when Langherans cells or DC are conjugated in vitrowith the hapten and then injected into a syngeneic organism (MacatoniaS. E., Immunology 59, 509-514 (1986)). The CB1 cells exposed to FITC orDNBS and then injected s.c. in native syngenic mice are able to induce aprimary response of CS type. The response is measured as swelling of themouse ear into which the cells have been injected; 10000 cells aresufficient to induce a CS response.

This response is antigen specific since untreated WO 94/28113PCTEP94iO1720 CB1 cells cannot generate induction. Therefore CB1 cellsare able to induce in vivo the activation of virgin T lymphocytes. Thecell lines of the invention have several advantages making themparticularly useful.

For example, CB1 cells can be used to stimulate ex vivo antigen specificT lymphocytes. Another example is the possibility to introduce thesecells into an organism in order to stimulate T lymphocytes in vivo. Theantigen can be administered contemporaneously or separately or, in thecase of pathogens, it may be already present in the organism. Anadditional advantage of CB1 cells is that the antigen can be loaded invitro before the introduction of the cells in vivo, thus allowing a muchmore limited amount of antigen or allowing the in vitro processing ofthe antigen without the need to inject it in vivo as a soluble protein.

A further advantage of these cell lines is that they can be used toinduce an immune response different from that obtained by injectingdirectly the antigen alone or in conduction with one or more adjuvant;in particular these cell lines can be used to induce T helperlymphocytes subsets such as the TH1 and TH2 subpopulations.

The molecular cloning of the provirus has the advantage to allowmodifications by genetic engineering and be used as a retroviral vector.

The cell lines of the invention may be used for the isolation ofcomponents or products which cannot be usually obtained because of thelimited proportion of APC, particularly DC, within the organs wherethese cells have been evidenced.

The following examples further illustrate the invention without limitingthe scope thereof.

The cells used in the examples are either commercially available orreported in scientific literature. The cells were generally cultivatedin RPMI, containing 2 mM β-mercaptoethanol, 10% heat-inactivated faetalcalf serum and kept at 37° C. in humidified atmosphere containing 5%Co₂.

Recombinant DNA Methods

When available, analytical grade reagents were used. If not otherwisestated, liquid and solid culture media were prepared according toManiatis T. et al., Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Lab. (1982), hereinafter referred to as "Maniatis".

Immunological Analysis Techniques

Analytical grade reagents were used. If not otherwise stated, the mediafor cell cultures, the buffer and washing solutions and the othermethods used were carried out according to Coligan J. E. et al.,"Current Protocols in Immunology" 1992 (J. Wiley and Sons Inc.; Media,Pa.-USA) hereinafter defined as "Coligan".

EXAMPLE 1

A genomic DNA library from N11 cell line in the pUC18 plasmid wasprepared according to known methods (Maniatis T. et al., MolecularCloning, a Laboratory Manual, Cold Spring Harbor Lab. (1982)). Methodsfor the library screening and for the probe preparation by nicktranslation are known and disclosed in Maniatis et al. The ligationproduct of the genomic DNA fragments generated by digestion with HindIIIand EcoRI and inserted into linearized pUC18 plasmid by the same methodsused to transform bacterial cells of the HB101 E. coli strain madecompetent for the incorporation of hexogenous genetic material(Maniatis).

The cells were then plated on solid LB-agar medium containing ampicillinat such a dilution so as to allow to obtain single colonies isolated onefrom the other.

After growth at 37° C. for 18 h the colonies were transferred ontonitrocellulose further (Schleicher and Schuell Co.). The filters weredried, washed and treated under vacuum at 70° C. (Maniatis). Thepre-hybridization, hybridization with a labelled probe derived from amyc-MH₂ gene fragment contained in the pMH2Hd plasmid were carried outaccording to Maniatis.

After suitable washings the filters were dried and exposed to X-rayfilms which are developed after 12-24 hours. After this screening, thecolonies found positive in an identical position on duplicate filterswere isolated and plated on LB-agar medium containing ampicillin so asto obtain colonies certainly deriving from a single transformed cell.

These colonies are then subjected to a second selection cycle identicalto that previously described.

Practically all colonies gave a positive signal in the second screeningcycle. One of these was chosed for further studies and named p316.

The insert was completely sequenced according to Sanger usingcommercially available reagents and the producer's instructions.

EXAMPLE 2

Preparation of a Line Producing the Retroviral Vector MIB-psi2-N11

The virus VN11 genome cloned into plasmid p316 was co-transfected withthe neo gene, derived from transposon TN5 and conferring resistance tothe neomycin analogue G418, into the cell line psi. This line is able toform infective viral particles when trasformed with an exogenous geneticmaterial containing informations in trans for the inclusion of the RNAproduced in the viral particle. The transformed clones were firstselected for the growth capacity in the presence of the G418 antibioticand subsequently for the presence of genomic RNA coded by the VN11provirus using a probe specific for the v-myc-MH2 gene. The so obtainedretroviral vector was named MIB-psi2-N11.

EXAMPLE 3

Generation of Dendritic Cell Lines

Spleen cell suspensions were prepared from newborn DBA/2 or BALB/C mice(Charles River, Italy) by lysing erythroid cells in ammonium chloride soas to remove erythrocytes according to known methods (Caligan). Thecells were then suspended in RPMI-1640 (Sigma) supplemented with 10% FCS(GIBCO), glutamine, penicillin, streptomycin and 0.5 mMβ-mercaptoethanol and plated at 10⁶ /ml density in 35 mm Petri dishes.Immortalization was carried out with the MIB-psi2-N11 retroviral vectorby filtering on 0.22 μsterile unit (Nalgene) the supernatant obtainedafter 24 hr subconfluent culture of the viral producer cell line diluted1:1 with complete medium containing 10 μg/ml polybrene (Sigma). After 1hr incubation at 37° C. in a 5% Co₂ incubator, half volume of freshmedium was added and then regularly changed twice a week. During thefirst week after infection, cells were fed with 10% L929.6C-conditionedmedia, reduced at 5% in the following two weeks, and then graduallyeliminated.

About 20-30 days after infection, multiple foci of dividing cells wereobserved in the Petri dishes. The cell line was considered establishedafter 20 passages, Once stabilized these lines were plated in 96 wellplates by limiting dilution and cloned.

EXAMPLE 4

Northern Blot Analysis

Northern blot analysis of mRNAs from CB1 cells and a positive controlwas carried out using a specific myc MH2 probe previously described(Proc. Natl. Acad. Sci. USA, 88, 7546, 1991). The myc MH2 3' probe wasderived from a chicken genomic library and it represents the 3' regionof the avian myc gene which does not cross-hybridize with the murine mycgenes.

EXAMPLE 5

Immunohistological Analysis of Dendritic Cell Lines

The clones CB1 and D2SC/1 were characterized in more detail by means ofantibodies specific for surface or intracellular markers of thedendritic cells such as the N418 anti, CD11c and anti B7 antibodies.These antibodies have been disclosed in the above reported referencesand were labelled with biotin using known methods (Coligan). Briefly 10⁶cells were incubated at room temperature for 15' in PBS containing 10%non-immune mouse or rat serum; this solution was then substituted withthe primary antibody labelled with biotin in PBS containing 0.1% w/v BSA(Sigma) and the sample was incubated for 30 at 4° C.; the cells werethen washed 3 times with PBS containing 0.1% w/v BSA (PBS/BSA) andincubated for 30' at 4° C. with a PBS/BSA solution containingStreptauidin/Phycoerythrin (Boehringer) according to the producer'sinstructions.

The cells were then analyzed by laser pulse flow cytofluorimetry(FACSort, Beckton & Dickinson); the dead cells were eliminated by meansof the data analysis pre-treating them with propidium iodide.

    __________________________________________________________________________    Comparison of published spleen Dendritic cell surface markers                 with the CB1 and D2SC/1 cell lines                                                     Crowley et                                                                          Vremec et                                                               al. 1989                                                                            al. 1992                                                                            Agger et                                                 ANTIGEN  (Steinman)                                                                          (Shortman)                                                                          al. 1990                                                                           D2SC/1                                                                              CB1                                           __________________________________________________________________________    MHCI     ++    ++++  +++  D +++ D +                                                                     K n.d.                                                                              K n.d.                                                                  L (AP+++)                                                                           L (AP+++)                                     MHC II   +++   ++++  +++  I-A ++                                                                              I-A +                                                                   I-E ++                                                                              I-E +                                         CD8α                                                                             -/++  ±/+++*                                                                           -/+* ±  ±                                          IL2-Rα                                                                           ±  -     ± -     -                                             CD28     n.d.  n.d.  n.d. ++    ++                                            B7       n.d.  n.d.  n.d. +     ++                                            CD2      n.d.  +     n.d. +     +                                             CD11a (LFA-1)                                                                          n.d.  n.d.  +    ++    ++                                            CD54 (ICAM-1)                                                                          n.d.  n.d.  n.d. +++   ++++                                          C11b (Mac-1)                                                                           +     +     +    n.d.  n.d.                                          CD11c (N418)                                                                           n.d.  n.d.  ++   ++    ++                                            α4 Integrin (R1-2)                                                               n.d.  n.d.  n.d. +++   ++                                            CD18     n.d.  n.d.  +++  +++   +++                                           CD44 (Pgp-1)                                                                           ++    ++++  n.d. ++++  ++++                                          HSA (J11D)                                                                             -/++* +++   -/+* +/+++*                                                                              +/+++*                                        B220     -     -     -    -     -                                             F4/80    ±  +     -    ++    +                                             CDw32 (Fcγ RII)                                                                  -     +     -    ++    +                                             FcεRI + II                                                                     n.d.  n.d.  n.d. -     -                                             __________________________________________________________________________     - negative                                                                    ± <10%                                                                     + 10-50%                                                                      ++ 50-90%                                                                     +++ >90%                                                                      ++++ 100%,mean >50x over background                                           n.d. not detected                                                             AP H2 L restricted antigen presentation                                       *two populations                                                              Agger R., Crowley M. T. and WitmerPack M.D. 1990, Intern. Rev. Immunol.       6:89-101, Crowley M., Inaba K., WitmerPack M. and Steinman R. M. 1989,        Cellular Immunology 118:108-125, Vremec D., Zorbas M., Scollay R.,            Saunders D. J., Ardavin C. F., Wu L. and Shortman K. 1992, J. Exp. Med.       176:47-58.                                                               

EXAMPLE 6

Immunohistochemical Analysis

For the analysis of intracellular markers, the CB1 cells were grown asabove on sterile glasses and then fixed in acetone for 2' at roomtemperature. The cells were then incubated with the first antibody for 1hour at room temperature in PBS containing 1% of non-immune mouse serum,washed three times with PBS/BSA and then incubated with peroxidaselabelled mouse antibodies against rat Igs (2A1 antibody) or againsthamster Igs (M342 antibody). The cells were then incubated in thepresence of diamino benzidine so as to evaluate the positivity thereof.

EXAMPLE 7

Analysis of the in Vitro Stimulatory Activity of CB1 Cells

The Mixed Lymphocyte Reaction (MLR) assay was carried out using a spleencell suspension obtained as described above from allogencic C57BL/6mice. CB1 dendritic cells or the macrophage cell line MT2/1 (P.Ricciardi-Castagnoli et al., 1992. Res. Immunol. 143, 101-106) were usedas stimulating cells pre-treating them with 25 μg/ml of Mitomycin C for20' at 37° C. in polystyrene test tubes. After washing, the stimulatingcells are washed with complete medium and plated on 96-well plates atdecreasing doses together with 30,000 cells/ml of T cells deriving fromC57BL/6 mice. The cells were co-cultured in the presence of completemedium containing 2 μCi/ml of 3H-TdR. T cells were obtained from thesplenic cell suspension by purification on nylon membrane. The cellswere incubated for 72 hours in the above reported conditions, filteredon filter-glass filter and the incorporated radioactivity was measuredby liquid scintillation counter (Betaplate, LKB-Wallac).

The presentation assay of the antigen was carried out adding decreasingdoses, starting from 0.5 μM, of whale sperm myoglobin (SpWMb) at 10000cells APC (CB1) preactivated with 100U/ml of IFN/γ or 200 μg/ml ofmrGM/CSF. The pre-activated APC were then co-cultivated with 10000 cellsof the murine T-T hybridoma 13.26.8 (obtained from Dr. A. Livingstone,Basel Institut for Immunology, Basel, CH) in flat-bottom, 96-wellsplates. After 24 hours of growth in Iscove (Sigma) medium containingantibiotics, glutamine and Beta-mercaptoethanol as above reported and 5%fetal bovine serum, 100 μl of supernatant from each well were trasferredto 96-well plates containing cells of the IL-2-dependent HT.2 line andassayed for the IL-2 content according to the described method (Coligan)using a calorimetric assay based on MTT (Sigma).

EXAMPLE 8

Analysis of the Capacity of CB1 Cells of Inducing T-Dependent Responsesin Vivo

CB1 cells were derivatized with 200 μg/ml of FITC (Sigma) or with 1mg/ml DNBS (2,4-dinitro benzene sulfonic acid, Eastman Kodak) for 30' at37° C. according to the method disclosed by Macatonia S. E. et al.,Immunology 59, 509-514, 10,000 cells were injected in a volume of 250 μlof HBSS (Sigma) subcutaneously in the back of syngeneic mice. After 5days, mice injected with derivatized cells or with non-derivatized cellsas a control, were treated with 25 μg of FITC or with 15 μg of DNFB(2,4-dinitro-1-fluorobenzene, Sigma) on both sides of each ear. The earthickness was measured with a micrometer immediately before treatmentand after 24, 48 and 72 hours.

EXAMPLE 9

Possibility of Obtaining MHC⁻ Variants of Dendriditic Cells DeletionMutants in the MHC Genes

Cultured cells were mutagenized by gamma rays with doses ranging from300 to 1000 rad from a cesium source and negatively selected withanti-MHC molecules type I an II antibodies and subsequent treatment withComplement.

According to the method disclosed by Moretta et al. (Proc. Natl. Acad.Sci. USA, 84, 1654-1658, 1987) it is possible to select, bycytofluorimetry, negative (or double-negative) variants for theexpression of MHC genes obtained after irradiation.

By this method, it was possible to obtain MHC⁻ (negative) dendritic celllines which can be subsequently transfected with the desired class I orclass II (MHC) genes. For instance, a plasmid containing the cDNA of theneo gene has been transfected into dendritic cells by means oflipofectin: to this purpose, 1×10⁶ cells were plated in 2 ml of culturemedium to which a solution containing 5 μg of DNA in 50 μl of culturemedium have been added. After 16 hours, further 2 ml of culture mediumwere added and the incubation continued for further 48 hours beforestarting the transfectants selection.

Utility

The present invention provides means for activating T-lymphocytes in anantigen-specific way both in vivo and in vitro.

For instance, it is possible to remove T cells from the organism,stimulating those specific for a given antigen and re-introducing themin the same organism so as to provoke an immune response to thatantigen. On the other hand, the cell lines of the invention may besensitized with a given antigen and then re-introduced into an MHCcompatible organism. In particular, this invention is useful in thosecases where the antigen purification is difficult or expensive, in thecases where the organism against which an immune response should beelicited is dangerous to handle or when vaccination with the sameorganism gives side-effects and moreover in those cases where it isusually difficult to elicit an effective and long-lasting immuneresponse.

I claim:
 1. Isolated immortalized mammalian dendritic cells, which have been immortalized by transfection with a vector containing a myc oncogene, wherein expression of the oncogene results in immortalization of the dendritic cells.
 2. The immortalized dendritic cells of claim 1, wherein the oncogene is a cellular oncogene.
 3. The immortalized dendritic cells of claim 1, wherein the oncogene is a viral oncogene.
 4. The immortalized dendritic cells of claim 1, wherein the vector is a retroviral vector and the oncogene is a viral oncogene.
 5. The immortalized dendritic cells of claim 4, wherein the myc oncogene is a v-myc oncogene, wherein said v-myc oncogene is fused to a gene encoding a retroviral glycoprotein.
 6. The immortalized dendritic cells of claim 1, wherein the dendritic cells were isolated, prior to immortalization, from a source selected from the group consisting of bone marrow, peripheral blood, Langerhans cells from the epidermis, follicular and interstitial dendritic cells from germinal centers and interdigitating dendritic cells from the lymphoid organs.
 7. The immortalized dendritic cells of claim 1, which retain the function of processing an antigen and presenting the processed antigen to T cells.
 8. A method of preparing the immortalized dendritic cells of claim 1, comprising transfecting mammalian dendritic cells with a vector containing a myc oncogene, wherein the oncogene is expressed in the transfected cells thereby immortalizing the cells.
 9. The immortalized dendritic cells of claim 1, which are primed with at least one antigen.
 10. A method of modulating T-cell function comprising contacting the immortalized dendritic cells of claim 9 with T cells.
 11. The method of claim 10, wherein the T cells are activated by the antigen-presenting immortalized dendritic cells.
 12. The method of claim 11, wherein the activation of the T cells is antigen-specific and MHC restricted. 